Biological systems and the processes operating therein, rest upon action of reciprocally interacting molecules. Molecular forces found in biological systems differentiate themselves from other molecular systems, especially in regard to chemical reactions and physical variations occurring within an entire system. Statements regarding biological, molecular reciprocation indicate that such systems should be analyzed and further investigations are to be carried out before more advanced comments about them are made.
For the investigation of measurements of molecular alternating action in biological systems, scanning probe microscopic equipment is employed in order to determine surface topographies with high lateral and vertical optical resolution. For example, a “lateral resolution” is a resolution in the plane of a surface of a biological system, which surface is under investigation. Similarly, a resolution vertically aligned thereto is designated as a “vertical resolution”.
Examples of scanning probe microscopy would encompass equipment such as scanning force microscopy (SFM) or atomic force spectroscopy (AFS).
In the case of atomic force microscopic equipment, besides the topography of a surface of a biological sample, the flexibility thereof and/or its inherent adhesive force is measured. Atomic force microscopy, in this case, is normally referred to as “Atomic Force Spectroscopy”. Atomic force spectroscopy detects molecular forces of a sample and does so with a probe which can directly contact the sample. Alternating action between interactive molecules can be quantitatively detected in this manner. A probe, supported on a cantilever, possesses a pointed tip. When engaged in an examination, a probe traverses over the surface of a sample, (or a sample is moved under a stationary probe) whereby the lateral and the vertical positions and/or linkages of the sample can be recorded and displayed.
Movements of the probe relative to the sample are enabled by the flexible characteristics of both the probe and its cantilever. On the basis of determined lateral and vertical positions and/or diversions of the sample, molecular forces acting on the sample are determined and therefrom, the topography of the sample is established.
Under usual procedure, movements of a sample, which are determined by optical instrumentation, detect resolutions of approximately 0.1 nm and simultaneously can detect forces of a few pN.
In order to determine surface topography or other characteristics of a biological sample, the surfaces of the sample and the probe of a scanning microscope are brought into contact with one another. The purpose of this is to determine that a force, acting between the two, lies within a predetermined range of, in some cases, 50-100 pN. Thereafter, the sample and the probe are laterally displaced relative to one another, so that a gridlike scan of the surface of the sample is carried out. Following this action, the sample and/or the probe are then separated vertically in order to maintain a predetermined force acting between them. Movements of the probe and the sample relative to one another can be carried out by an appropriate device, which employs a piezo-ceramic actuator.
High resolutions of position or force are very sensitive to variation when their operating equipment is exposed to external disturbances, in one instance, ambient thermal effects can lead to bending of the cantilever. Since the degree of displacement of the cantilever can typically be caused by laser beam variance or by thermal or surface effects, such bending of the cantilever leads to a correction value which is then applied to the measured values. Thermal effects, also cause a variation in the separating distance between the probe and the sample, whereby the force between the probe and the sample is altered. Thermal variations are typically made slowly and occupy long time periods. Therefor, such variations are commonly known as “drifts”. Such drifts are based also on other influences, including thermally induced, substantial positional variations in adjustment means, or variances in the probe itself, or in holding devices or in securement means or yet in sample carriers. Such interfering variances, which lead to hysteresis characteristics and/or to creep, are detected by piezo-ceramic units as these are found in the said actuator. The stated unwanted variances could result in cumulative error, which can alter the distance between the probe and the sample.
In the area of imaging atomic force microscopy and atomic force microscopy, up to the present, drifts in the course of the beam were intended to be compensated for by manual intervention. This was done when, the user observed from the value of the position detector that the cantilever was no longer in contact with the sample. A correction of the beam path was made by a manual adjustment of mechanical settings on an instrument board. In this way, the measuring laser beam was caused to fall once again on the desired target on the position detector.
In U.S. Pat. No. 5,077,473 is disclosed a method for compensation of drift in the x and/or y directions of a positioning apparatus for a sample. The positioning apparatus is driven by a piezo-actuator which is subject to drift signals in the x and/or y direction. This drift is compensated for, since to a control signal directed to the positioning apparatus an auxiliary signal is added, which compensates for the drift in the x and/or y directions.
The purpose of the invention is to make available an improved method for scanning force microscopy as well as for atomic force spectroscopy.